The goals of the proposed research are to isolate a small, genetically defined segment of a eucaryotic genome and to determine the role of eucaryotic RNA polymerase in selective transcription of a specific gene in vitro. The genes which code for glycolytic enzymes in yeast have been chose as a model for these studies. A small segment of the yeast genome containing a glycolytic gene will be isolated by 1) digestion of a total yeast DNA with a restriction endonuclease, 2) ligation of these unique DNA fragments to a bacterial plasmid DNA, 3) propagation of these recombinants in bacterial cells, 4) selection of transformants containing the glycolytic gene by molecular hybridization of transformant plasmid DNA with complementary DNA (cDNA) synthesized in vitro from purified glycolytic messenger RNA (mRNA) with reverse transcriptase. Messenger RNAs coding for specific glycolytic enzymes will be isolated by poly(U)-cellulose affinity chromatography, sucrose density gradient centrifugation, and polyacrylamide gel electrophoresis. Mapping of the glycolytic gene within the yeast fragment of the recombinant DNA will be carried out by hybridization analysis of subfragments of this yeast DNA fragment, generated by additional site-specific endonucleases with the cDNA and purified mRNA. Recombinant DNA containing a specific glycolytic gene will be isolated intact and will serve as a template for purified yeast RNA polymerases. Strand-specific initiation, as well as selective initiation and termination of RNA synthesis will be investigated. The aim of these studies is the eventual reconstitution of selective transcription of a specific eucaryotic gene in vitro.